We previously demonstrated the oncogenic potential of Stat5, with thymic lymphoblastic lymphomas developing in a significant proportion of transgenic (TG) mice over-expressing Stat5a or Stat5b in lymphocytes1. With our current Merit Review funding we investigated the mechanism of Stat5-mediated lymphomagenesis by exploring the contributions of major histocompatibility complex (MHC)/T-cell receptor (TCR) and pre-TCR signals. We demonstrated that Stat5b TG mice unexpectedly develop CD8+ lymphoma in the absence of either pre-TCR signaling or normal thymic MHC/TCR signaling. Indeed, acceleration of Stat5b transgene-mediated lymphoma occurred on TCR1-/- and pre-TCR1-/- backgrounds2. These data support our hypothesis: alterations in T-cell development cooperate with cytokine/growth factor-mediated pathways in immature thymocytes to give rise to lymphoblastic T-cell lymphomas in Stat5b TG mice. In this proposal we will extend these studies to explore the signaling mechanisms and genes involved in transformation of immature thymocytes in lymphoblastic lymphoma. Importantly, we have demonstrated the ability of Jak and calcineurin inhibitors to decrease proliferation of Stat5b TG thymocytes in vitro. Inhibition of signaling pathways including the Jak/Stat5 pathway provides a novel therapeutic approach to lymphoma. Aim 1: Test the hypothesis that cytokine/growth factor-mediated transformation occurs at or prior to the immature single positive (ISP) stage. To expand our understanding of Stat5b-mediated lymphomagenesis, we will determine the site of transformation. This will allow us to examine phenotypic changes and gene expression associated with transformation. Moreover, we will compare the effects of hematopoietic growth factors and cytokines on Stat5b TG and WT immature populations. Sub-Aim 1: Define the population and developmental stage of lymphoma cells. We will enrich immature thymic and BM populations using a combination of magnetic separation and flow cytometry. These populations will be examined for surface expression of phenotypic markers as well as gene expression of lineage specific genes. Growth of subcutaneously (s.q.) injected cells will define the malignant subpopulations. Subaim 2: Define requirements for growth and development of immature thymic precursors in Stat5b TG mice. Enriched immature populations will be cultured with stromal cells expressing the Notch delta like ligand (OP9-DL1) along with cytokines (IL-7), and growth factors (stem cell factor [SCF], fms-like tyrosine- kinase 3 [Flt3] ligand). We will examine cell growth, development, and phenotypic changes over time. Aim 2. Test the hypothesis that IL-7/Jak signaling contributes to Stat5-mediated lymphomas. To clarify the role of IL-7 signaling in Stat5b-mediated lymphoma we will cross Stat5b TG mice onto IL-7R1Y449 mice. These mice cannot signal through Stat5 but have preserved T-cell development. Aim 3. Inhibit Stat5b TG lymphoma in vivo. Our data demonstrate that signals downstream of TCR and cytokine receptors promote lymphoma cell proliferation. Moreover, Stat5 up-regulates anti-apoptotic Bcl-2 family members. Thus, we propose to use a combination of therapies targeting cytokine (CP-690550), calcineurin (Cyclosporin A) and Bcl-2 family members (apogossypol). With these studies we will better define the timing and stage of transformation. We will determine the contribution of Notch1, growth factors and cytokine signaling to the process. Using multiple targeted therapies based on evolving understanding of lymphomagenesis, we will inhibit Stat5-mediated lymphoma in vivo. These experiments have the potential to advance the therapeutic approaches in lymphoma and other malignancies. PUBLIC HEALTH RELEVANCE: Stat5 is associated with a wide variety of human malignancies, including non-Hodgkin's lymphoma (NHL). NHL is the sixth most common cancer in the United States with 66,120 estimated new cases and 19,160 deaths from NHL in 2008. Moreover, Hodgkin's and non-Hodgkin's lymphoma are two of the ten diseases and conditions recognized by the VA as service-connected for Vietnam Veterans, based on their exposure to Agent Orange. We propose to analyze the mechanisms by which Stat5 mediated signaling results in lymphoma, and to determine the requirement for Jak/Stat5 signaling in the maintenance of lymphoma cell survival. Using these mechanistic insights we will inhibit Stat5b TG-mediated lymphoma in vivo. These studies will provide insights into the biology of lymphoma, and potentially lead to new therapies for this disease.